This invention relates generally to molecular cloning and, more specifically, to ligating polynucleotides.
The techniques of molecular cloning are used in virtually all realms of scientific research, from anthropology to forensic science to drug discovery. The role of molecular cloning in scientific research is expanding into new territory and increasing in importance, especially as techniques of molecular cloning are becoming easier to access and perform. The sequencing of the human genome is a prominent example of the contribution of molecular cloning to both basic science and medical research. The techniques of molecular cloning will also be central in the next phase of the human genome project, uncovering the functions of hundreds of newly identified human genes. This phase of the project will yield insights into the underlying causes of human disease, resulting in improved diagnostics and therapeutics. Increased efficiency in molecular cloning methods will accelerate progress in this, as well as many other areas of scientific endeavor.
Ligation of polynucleotides is a key step in the process of molecular cloning. Ligation is the joining of two nucleic acid molecules by the formation of a phosphodiester bond, which is the naturally occurring linkage between the nucleotides that make up a nucleic acid molecule. The ligation step in a molecular cloning procedure is typically performed using a ligase enzyme. This step is generally time-consuming because ligase enzymes can require incubation times of several hours to achieve optimal reaction efficiency. In recent years, a ligation method that works without a ligase enzyme has been developed and commercialized. The method employs a sequence specific viral topoisomerase enzyme. Although generally more time efficient, the “topo cloning” method is less flexible than standard cloning methods. For example, the method requires that a special sequence be incorporated into the DNA to be cloned. Further, “topo cloning” limits the user to a particular cloning site and therefore cannot be used for direct cloning of DNAs into user-selected restriction sites.
Thus, there exists a need for improved methods for ligating nucleic acids. The present invention satisfies this need and provides related advantages as well.